Therapeutic use of chardonnay seed products

ABSTRACT

The present disclosure relates to health benefits of Chardonnay seed products.

1. BACKGROUND

1.1. Metabolic Conditions

Obesity is a well-known risk factor for the development of many verycommon diseases such as atherosclerosis, hypertension, type 2 diabetes(non-insulin dependent diabetes mellitus (NIDDM)), dyslipidemia,coronary heart disease, and osteoarthritis and various malignancies. Italso causes considerable problems through reduced motility and decreasedquality of life. The incidence of obesity and thereby also thesediseases is increasing throughout the entire industrialized world.

The term obesity implies an excess of adipose tissue. In this contextobesity is best viewed as any degree of excess adiposity that imparts ahealth risk. The cut off between normal and obese individuals can onlybe approximated, but the health risk imparted by the obesity is probablya continuum with increasing adiposity.

Even mild obesity increases the risk for premature death and conditionssuch as diabetes, dyslipidemia, hypertension, atherosclerosis,gallbladder disease and certain types of cancer. In the industrializedwestern world the prevalence of obesity has increased significantly inthe past few decades. Because of the high prevalence of obesity and itshealth consequences, its prevention and treatment should be a highpublic health priority.

When energy intake exceeds expenditure, the excess calories are storedpredominately in adipose tissue, and if this net positive balance isprolonged, obesity results, i.e., there are two components to weightbalance, and an abnormality on either side (intake or expenditure) canlead to obesity. This process may be counteracted by increasing theenergy expenditure (for instance via exercise) or decreasing the energyintake (for instance by dieting). Except for exercise, diet and foodrestriction, which is not feasible for a vast number of subjects, noconvincing treatment for reducing body weight effectively and acceptablycurrently exist.

One possible way to increase energy expenditure is by increasing themetabolic rate. Agents which act by increasing the metabolic rate maythus be useful for treating obesity, but also for treating otherconditions such as atherosclerosis, hypertension, diabetes, especiallytype 2 diabetes (NIDDM (non-insulin dependent diabetes mellitus)),dyslipidemia, coronary heart disease, gallbladder disease,osteoarthritis and various types of cancer such as endometrial, breast,prostate and colon cancers and the risk for premature death.

Thus, it would be desirable to identify agents that can increase energyexpenditure. Preferably, such agents would be natural agents that avoidside effects associated with pharmaceutical compounds.

1.2. Gut Biome

The infant gut is sterile before birth. After birth, the gut is rapidlycolonized by environmental bacteria until a dense gut biome isestablished. Infants delivered vaginally acquire gut colonizing bacteriafrom their mother's vaginal and fecal flora. In contrast, infantsdelivered by cesarean section are not exposed to their mother's vaginaland fecal flora during birth and thus develop a gut biome that isdifferent in composition than the gut biome of infants deliveredvaginally. These differences in gut biome composition persist in themonths immediately following birth. Likewise, differences in gut biomecomposition have been observed between infants that are breast fed andthose that are formula fed.

The adult human gut has approximately 10¹³ (10,000,000,000,000)individual residents (Bäckhead, F., et al. (2004) PNAS Volume 101; no 44pp. 15718-15723) and there are three consistent enterotypes establishedacross many human cultural backgrounds (Arumugam, M. et al. (2011)Nature Vol. 473 pp. 174-180). The gut biome is influenced by plant basedpolyphenols in the diet and it is believed that the microbes convertthem to be bioavailable to the human host (Rastmanesh, R. (2011),Chemico-Biol. Interact. Vol. 189 pp. 1-8; Moco, S., F. J. Martin, and S.Rezzi. (2012) J. Proteome Res. Volume 11, pp. 4781-4790). Acting as anorgan, the gut biome is also responsible for conversion and productionof key vitamins such as cholecalciferol (vitamin D₂₅), biotin (vitaminH), riboflavin (vitamin B₂), pantothenate (vitamin B₅), ascorbate(vitamin C), thiamine (vitamin B₁) and folate (vitamin B₉); particularlyin two (Bacteroides and Prevotella) of the three enterotypes(Bacteroides, Ruminococcus, and Prevotella) discovered. Some polyphenolshave been described as vitamins in their own right by Dr. NormanHollenberg, Professor of Medicine at Harvard Medical School. However,modern diets, particularly Western diets comprising high amounts ofprocessed foods, may promote a gut biome composition that fails toconvert or produce adequate or optimal amounts of these key vitamins.

Thus, it would be desirable to identify agents that can “fill in thenutritional gaps” caused by consumption of traditionally processed foodsand modulate the human gut biome to achieve improved health. Preferably,such agents would be natural agents that avoid side effects associatedwith pharmaceutical compounds.

2. SUMMARY

The present disclosure relates to health benefits of Chardonnay seedproducts.

In certain aspects, the present disclosure relates to a method ofincreasing lipid metabolism in a mammal by administering an amount ofChardonnay seed product that is effective to increase lipid metabolismin the mammal.

In certain aspects, the present disclosure relates to a method ofincreasing a mammal's metabolic rate by administering an amount ofChardonnay seed product that is effective to increase the mammal'smetabolic rate.

The present disclosure also relates a method of treating or preventingobesity, a cardiovascular disease, a dyslipidemia, a dyslipoproteinemiaor a glucose metabolism disorder in a mammal by administering to amammal an amount of Chardonnay seed product that is effective to treator prevent obesity in the mammal.

The present disclosure further relates to a method of treating orpreventing a cardiovascular disease in a mammal by administering to amammal an amount of Chardonnay seed product that is effective to treator prevent said cardiovascular disease in the mammal. In certainaspects, the cardiovascular disease is arteriosclerosis,atherosclerosis, stroke, ischemia, endothelium dysfunctions, peripheralvascular disease, coronary heart disease, myocardial infarcation,cerebral infarction or restenosis.

The present disclosure further relates to a method of treating orpreventing a dyslipidemia in a mammal by administering to a mammal anamount of Chardonnay seed product effective to treat or prevent saiddyslipidemia in the mammal. In certain aspects, the dyslipidemia ishyperlipidemia or low blood levels of high density lipoprotein (HDL)cholesterol. In certain aspects, the hyperlipidemia is familialhypercholesterolemia, familial combined hyperlipidemia, reduced ordeficient lipoprotein lipase levels or activity, hypertriglyceridemia,hypercholesterolemia, high blood levels of ketone bodies, high bloodlevels of Lp(a) cholesterol, high blood levels of low densitylipoprotein (LDL) cholesterol, high blood levels of very low densitylipoprotein (VLDL) cholesterol, or high blood levels of non-esterifiedfatty acids.

The present disclosure further relates to a method of treating orpreventing a dyslipoproteinemia in a mammal by administering an amountof Chardonnay seed product effective to treat or prevent saiddyslipoproteinemia. In certain aspects, the dyslipoproteinemia is highblood levels of LDL, high blood levels of apolipoprotein B (apo B), highblood levels of Lp(a), high blood levels of apo(a), high blood levels ofVLDL, low blood levels of HDL, reduced or deficient lipoprotein lipaselevels or activity, hypoalphalipoproteinemia, lipoprotein abnormalitiesassociated with diabetes, lipoprotein abnormalities associated withobesity, lipoprotein abnormalities associated with Alzheimer's Disease,or familial combined hyperlipidemia.

The present disclosure further relates to a method of treating orpreventing a glucose metabolism disorder in a mammal by administering toa mammal an amount of Chardonnay seed product effective to treat orprevent said glucose metabolism disorder in the mammal. In certainaspects, the glucose metabolism disorder is impaired glucose tolerance,insulin resistance, insulin resistance related breast, colon or prostatecancer, diabetes, pancreatitis, hypertension, polycystic ovariandisease, high levels of blood insulin, or high levels of blood glucose.In certain aspects, the diabetes is non-insulin dependent diabetesmellitus (NIDDM), insulin dependent diabetes mellitus (IDDM),gestational diabetes mellitus (GDM), or maturity onset diabetes of theyoung (MODY).

The disclosure further relates to a method of treating or preventingmetabolic syndrome in a mammal by administering to a mammal an amount ofChardonnay seed product effective to treat or prevent metabolic syndromein the mammal.

In certain aspects, an amount is administered that is effective tomodulate expression of one or more genes involved in fat, cholesterol,and/or bile metabolism. In specific embodiments, the amount is effectiveto increase expression of ACOX1 in hepatic tissue, to increaseexpression of CYP51 in hepatic tissue, to increase expression of CYP7a1in hepatic tissue, to decrease expression of SCD1 in hepatic tissue,and/or to decrease expression of ABCG5 in hepatic tissue, for example byat least 10%, at least 20%, at least 50%, or at least 100%.

The present disclosure further relates to a method of increasing theamount of Clostridium bacteria in the gut of a mammal by administeringto the mammal an amount of Chardonnay seed product effective to increasethe amount of Clostridium bacteria in the gut of the mammal.

The present disclosure further relates to a method of decreasing theamount of Enterobacteriaceae bacteria in the gut of a mammal byadministering to the mammal an amount of Chardonnay seed producteffective to decrease the amount of Enterobacteriaceae bacteria in thegut of the mammal.

The present disclosure further relates to a method of increasing theamount of Bacteroides fragilis group bacteria in the gut of a mammal byadministering to the mammal an amount of Chardonnay seed producteffective to increase the amount of Bacteroides fragilis group bacteriain the gut of the mammal.

The present disclosure further relates to a method of treating orpreventing lactic acidosis in a mammal by administering to the mammal anamount of Chardonnay seed product effective to treat or prevent lacticacidosis.

In certain embodiments of the methods, a second grape seed or grape skinproduct which is not a Chardonnay seed product is administered to themammal. In certain aspects, the combination of Chardonnay seed productand second grape seed or grape skin product provides a therapeuticeffect or health benefit which is greater than the effect ofadministration of Chardonnay seed product alone.

In certain aspects, the Chardonnay seed product is prepared from seedshaving an epicatechin content of at least 600 mg of epicatechin per 100g of seeds or an epicatechin content of at least 700 mg of epicatechinper 100 g of seeds. In specific embodiments, the epicatechin contentranges from 600-800 mg/100 g of seeds or from 650-800 mg/100 g of seeds.

In certain embodiments, the Chardonnay seed product is incorporated intoa food or beverage product.

In certain embodiments, the Chardonnay seed product is Chardonnay seedflour. In certain embodiments, the Chardonnay seed product is Chardonnayseed extract. In certain embodiments, the Chardonnay seed product isfrom grapes grown in a Winkler region climate type I, II, III or IV.

In certain embodiments, the mammal is a domestic pet, e.g., a cat or adog. In other embodiments of the methods, the mammal is a human. Inother embodiments, the human is an infant.

The present disclosure further relates to an infant formula comprising aChardonnay seed product and methods of using the infant formula topromote gut microbiome development in an infant. In certain aspects, theinfant formula promotes gut microbiota development when administered toan infant by modulating levels of gut bacteria. In certain embodiments,the infant was delivered by cesarean section. In certain embodiments,the infant is formula fed.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Protein, fat, ash, carbohydrate, and total dietary fiber contentof Chardonnay (Chr), Cabernet (Cab), and Syrah (Syr) seeds (Sd) andskins (Sk).

FIG. 2: Dietary composition of hamsters in Example 1.

FIG. 3: Seed diets body weight by week of the animals of Example 1.

FIG. 4: Skin diets body weight by week of the animals of Example 1.

FIG. 5: Weekly feed intake of the animals of Example 1.

FIG. 6: Total feed intake of the animals of Example 1.

FIG. 7: Total caloric intake of the animals of Example 1.

FIG. 8: Plasma lipoprotein cholesterol levels of the animals of Example1 at the end of four weeks.

FIG. 9: Blood glucose levels of the animals of Example 1 at the end offour weeks.

FIG. 10: Organ weights of the animals of Example 1 at the end of fourweeks.

FIG. 11: Epicatechin levels in the seed and skin of different grapevarieties.

FIG. 12: Body weight, weight gain, and total feed intake of animals ofExample 2 fed diets comprising 3%, 7%, and 10% Chardonnay seed flour byweight.

FIG. 13: VLDL, LDL, and HDL cholesterol levels of animals of Example 2fed Chardonnay seed ethanol extract (40EtChrSdEx), Chardonnay seedmethanol extract (MeChrSdEx), Chardonnay seed ethanol extract residue(40EtChrSdRes), Chardonnay seed methanol extract residue (MeChrSdRes),Chardonnay seed flour at 10%, 7%, and 3% by diet weight (10% ChrSd, 7%ChrSd, and 3% ChrSd, respectively), 10% White Riesling seed flour bydiet weight (10% WRSd), and 10% Sauvignon Blanc seed flour by dietweight (10% SBSd) at the end of four weeks.

FIG. 14: LDL/HDL cholesterol ratio of animals of Example 2 fedChardonnay seed ethanol extract (40EtChrSdEx), Chardonnay seed methanolextract (MeChrSdEx), Chardonnay seed ethanol extract residue(40EtChrSdRes), Chardonnay seed methanol extract residue (MeChrSdRes),Chardonnay seed flour at 10%, 7%, and 3% by diet weight (10% ChrSd, 7%ChrSd, and 3% ChrSd, respectively), 10% White Riesling seed flour bydiet weight (10% WRSd), and 10% Sauvignon Blanc seed flour by dietweight (10% SBSd) at the end of four weeks.

FIG. 15: Liver, Epidydimal Adipose (EA), and Retroperitoneal Adipose(RA) weights of animals of Example 2 fed Chardonnay seed ethanol extract(40EtChrSdEx), Chardonnay seed methanol extract (MeChrSdEx), Chardonnayseed ethanol extract residue (40EtChrSdRes), Chardonnay seed methanolextract residue (MeChrSdRes), Chardonnay seed flour at 10%, 7%, and 3%by diet weight (10% ChrSd, 7% ChrSd, and 3% ChrSd, respectively), 10%White Riesling seed flour by diet weight (10% WRSd), and 10% SauvignonBlanc seed flour by diet weight (10% SBSd) at the end of four weeks.

FIG. 16: Relative adipose gene expression of Example 3.

FIG. 17: Relative hepatic gene expression of Example 3.

FIG. 18: Total plasma lipoprotein cholesterol levels of animals ofExample 4 fed diets supplemented with Chardonnay seed flour (CharSdFl),Vitacost® grape seed extract (VC1 and VC7), Mega Natural® BP grape seedextract (BP1, BP7), Leucoselect® grape seed extract (L1, L7), catechin(Cat), and epicatechin (EpiCat) at the end of four weeks.

FIG. 19: VLDL, LDL, and HDL cholesterol levels of animals of Example 4fed diets supplemented with Chardonnay seed flour (CharSdFl), Vitacost®grape seed extract (VC1 and VC7), Mega Natural® BP grape seed extract(BP1, BP7), Leucoselect® grape seed extract (L1, L7), catechin (Cat),and epicatechin (EpiCat) at the end of four weeks.

FIG. 20: Total fecal bacteria levels of animals of Example 5 fed acontrol diet, high fat diet supplemented with Cabernet seed flour(RGF+HF), high fat diet supplemented with Chardonnay seed flour(WGF+HF), and high fat control diet (HF).

FIG. 21: Enterococcus spp. levels of animals of Example 5 fed a controldiet, high fat diet supplemented with Cabernet seed flour (RGF+HF), highfat diet supplemented with Chardonnay seed flour (WGF+HF), and high fatcontrol diet (HF).

FIG. 22: Bifidobacterium spp. levels of animals of Example 5 fed acontrol diet, high fat diet supplemented with Cabernet seed flour(RGF+HF), high fat diet supplemented with Chardonnay seed flour(WGF+HF), and high fat control diet (HF).

FIG. 23: Lactobacillus spp. levels of animals of Example 5 fed a controldiet, high fat diet supplemented with Cabernet seed flour (RGF+HF), highfat diet supplemented with Chardonnay seed flour (WGF+HF), and high fatcontrol diet (HF).

FIG. 24: Clostridium Cluster IV bacteria levels of animals of Example 5fed a control diet, high fat diet supplemented with Cabernet seed flour(RGF+HF), high fat diet supplemented with Chardonnay seed flour(WGF+HF), and high fat control diet (HF).

FIG. 25: Bacteroides fragilis group bacteria levels of animals ofExample 5 fed a control diet, high fat diet supplemented with Cabernetseed flour (RGF+HF), high fat diet supplemented with Chardonnay seedflour (WGF+HF), and high fat control diet (HF).

FIG. 26: Enterobacteriaceae levels of animals of Example 5 fed a controldiet, high fat diet supplemented with Cabernet seed flour (RGF+HF), highfat diet supplemented with Chardonnay seed flour (WGF+HF), and high fatcontrol diet (HF).

4. DETAILED DESCRIPTION

4.1. Chardonnay Seed Products

The present invention relates to Chardonnay seed products and methods oftreating or preventing obesity and other conditions using Chardonnayseed products. Preferably, the Chardonnay seed products are producedfrom grapes grown in Winkler region climate types I-IV (Jones et al.,2010, Am. J. Enol. Vitic. 61(3):313-326). In some embodiments, theChardonnay seed products are produced from grapes grown in the coastalvalleys of Northern California, e.g., the Napa Valley and/or the SonomaValley. In another embodiment, the Chardonnay seed products are producedfrom grapes grown in other, hotter, inland valley vineyards, e.g., inWinkler region climate types IV-V. In a preferred embodiment, theChardonnay seed product is from grapes grown in a coastal region.

In an embodiment, the Chardonnay seed products contain the defattedportion of Chardonnay seed, such as Chardonnay pomace meal, Chardonnaypomace flour, Chardonnay seed meal, or, most preferably, Chardonnay seedflour. In some embodiments, the Chardonnay seed product includes thecontents of defatted Chardonnay seeds that are not extractable by anorganic solvent, e.g., are not extractable by ethanol and/or methanol.In certain aspects, the Chardonnay seed product is prepared from seedshaving an epicatechin content of at least 600 mg of epicatechin per 100g of seeds or an epicatechin content of at least 700 mg of epicatechinper 100 g of seeds. In specific embodiments, the epicatechin contentranges from 600-800 mg/100 g of seeds or from 650-800 mg/100 g of seeds.

As used herein, “Chardonnay seed meal” is ground whole seeds and“Chardonnay seed flour” is ground seed after the oil has been extracted.Chardonnay seed flour may be obtained using the “cold press”, “hotpress” and solvent extraction processes as are known in the art toextract the oil from seeds yielding defatted seed flour. The meal orflour can be dried to the desired moisture content using conventionaldrying techniques suitable for drying food products.

The dried meal or flour is further ground under ambient temperatureconditions to form Chardonnay seed powder having free-flowing particles.In an embodiment, the free-flowing particles can range from a size notexceeding 841 microns (20 mesh) to a size not exceeding 37 microns (400mesh). In certain embodiments, the size does not exceed 20 mesh, 40mesh, 60 mesh, 80 mesh, 100 mesh, 200 mesh, 300 mesh, or 400 mesh.

In an exemplary method, Chardonnay seed flour is made by separating anddrying Chardonnay grape seeds, for example from the pomace producedafter Chardonnay grapes are pressed to produce grape juice (e.g., tomake wine). The grape seeds can be “cold-pressed” to defat them(producing Chardonnay seed oil as a byproduct). Grape seed flours aremilled from the press cake after the oil is expelled. In one embodiment,after juicing the grape the seed is separated from the skins, cleaned,mechanically defatted, finely milled and sifted to create an 100 mesh(150 micron) flowable powder.

Chardonnay seed flour can also be purchased from Apres Vin (Yakima,Wash.), Botanical Oil Innovations (Spooner, Wis.) or Fruitsmart, Inc.(Grandview, Wash.). The FruitSmart Chardonnay seed flour is an 85 meshflowable powder but can be further milled and sifted to produce a flourwith a smaller particle size.

In an embodiment, skins, stems and leaves (the remainder of pomace) areremoved from the seeds prior to pressing. Removal of the skins, stems,and leaves allows for optimal oil pressing.

“Chardonnay seed extract” is made by solvent extraction of Chardonnayseeds with a suitable solvent, such as ethanol or methanol. For example,“40EtChrSdEx” is a Chardonnay seed extract made using a 40% ethanolsolution as the extraction solvent. The extraction process, in additionto the extract containing the solvent soluble components, also producesa residue of non-soluble solids.

4.2. Effective Amount

In an embodiment of the methods of the invention, the amount ofChardonnay seed flour consumed as a percentage of daily diet is at least3%, at least 5%, or at least 8% by mass. Preferably, 5-10%, morepreferably 7%, and in some embodiments 10% of the daily diet by mass isChardonnay seed flour.

In an embodiment of the methods of the invention, the amount ofChardonnay seed flour consumed as a percentage of daily diet is at least3%, at least 5%, or at least 8% of total calories consumed. Preferably,5-10%, more preferably 7%, and in some embodiments 10% of the dailycalories are from Chardonnay seed flour.

In another embodiment, the amount of Chardonnay seed flour consumeddaily is at least 10 g, at least 15 g, at least 20 g, at least 25 g, atleast 30 g, at least 35 g, at least 40 g, or at least 45 g. Preferably,50 g of Chardonnay seed flour is consumed daily.

In another embodiment, the amount of Chardonnay seed flour consumeddaily is at least 1 tablespoon, at least 2 tablespoons, at least 3tablespoons, at least 4 tablespoons, or at least 5 tablespoons.

In another embodiment, the amount of Chardonnay seed flour consumeddaily on a Chardonnay seed flour weight:body weight basis is at least0.2 g/kg, at least 0.5 g/kg, or at least 0.7 g/kg. Preferably at least 1g of Chardonnay seed flour per kg of body weight is consumed per day.

In certain aspects, an amount of Chardonnay seed flour is administeredthat is effective to modulate expression of one or more genes involvedin fat, cholesterol, and/or bile metabolism. In specific embodiments,the amount is effective to increase expression of ACOX1 in hepatictissue, to increase expression of CYP51 in hepatic tissue, to increaseexpression of CYP7a1 in hepatic tissue, to decrease expression of SCD1in hepatic tissue, and/or to decrease expression of ABCG5 in hepatictissue, for example by at least 10%, at least 20%, at least 50%, or atleast 100%.

Chardonnay seed flour can be substituted with Chardonnay pomace meal,pomace flour, skin flour, seed extract, or seed meal in the methods ofthe invention. The amount of Chardonnay pomace meal, pomace flour, skinflour, seed extract, or seed meal that will need to be consumed daily toattain the same benefit as a given amount of Chardonnay seed flour canreadily be determined by those skilled in the art. For example, it isexpected that a subject will need to consume about three times as muchChardonnay skin flour to achieve the same benefit as a given amount ofChardonnay seed flour.

In certain embodiments, the Chardonnay seed product, e.g., Chardonnayseed flour, is taken at least twice a week, at least 3 times a week, orevery other day. Preferably, the Chardonnay seed product is incorporatedinto the daily diet.

The Chardonnay seed product, e.g., Chardonnay seed flour, can be takenfor an amount of time sufficient to treat and/or prevent a conditionamenable to treatment and/or prevention by Chardonnay seed product asdescribed herein. The Chardonnay seed product a can be taken for atleast one week, at least 2 weeks, at least 3 weeks, at least one month,at least 2 months, at least 3 months, at least 6 months, at least ayear, or indefinitely.

In certain embodiments of the methods, a second grape seed or grape skinproduct which is not a Chardonnay seed product is administered to themammal. In certain aspects, the combination of Chardonnay seed productand second grape seed or grape skin product provides a therapeuticeffect or health benefit which is greater than the effect ofadministration of Chardonnay seed product alone.

In other embodiments, the amount of Chardonnay seed product and amountof the second grape seed or grape skin product are selected so that theeffect achieved is at least the same as the effect achieved by a givenamount of Chardonnay seed product administered alone.

4.3. Chardonnay Seed Compositions

The Chardonnay seed products can be included in a variety of foodproducts, such as nutritional beverages (e.g., nutritional shakes),baked goods (e.g., cookies, brownies, cake, breads, biscuits, crackers),puddings, confections (i.e., candy), snack foods (e.g., pretzels), icecream, frozen confections and novelties, or non-baked, extruded foodproducts such as bars, including health or energy bars. The Chardonnayseed product can also be provided as a nutritional supplement, either intablet form or as a powder for use as a nutritional food additive.

In one embodiment, the Chardonnay seed product can be blended with otherdry food materials for use in the preparation of food products enrichedwith Chardonnay seed products. Dry food materials include, for example,dry starch-containing materials, dry protein-containing materials orcombinations thereof. Suitable starch-containing materials may bederived from, for example, rice, corn, soybeans, sunflower, canola,wheat, oats, rye, potato, or any combination thereof. Suitable dryprotein-containing materials may be derived from for example, meat,milk, fish or any combination thereof. For baking applications, theChardonnay seed product is suitable used in an amount ranging from 3% to15% of the dry food material (e.g., white or whole wheat flour). The dryfood may optionally also include additional ingredients such asvitamins, mineral fortifiers, salts, colors, flavors, flavor enhancersor sweeteners.

Chardonnay seed products can be incorporated into beverages, processedmeats, frozen desserts, confectionery products, dairy-type products,sauce compositions, and cereal grain products. Beverage productsinclude, for example, smoothies, infant formula, fruit juice beverages,yogurt beverages, coffee beverages, beer, dry beverage mixes, tea fusionbeverages, sports beverages, soy liquors, soda, slushes, and frozenbeverage mixes. Meat products include, for example, ground chickenproducts, water-added ham products, bologna, hot dogs, franks, chickenpatties, chicken nuggets, beef patties, fish patties, surimi, bacon,luncheon meat, sandwich fillings, deli meats, meat snacks, meatballs,jerky, fajitas, bacon bits, injected meats, and bratwurst. Confectioneryproducts include, for example, chocolates, mousses, chocolate coatings,yogurt coatings, cocoa, frostings, candies, energy bars, and candy bars.Frozen dessert products include, for example, ice cream, malts, shakes,popsicles, sorbets, and frozen pudding products. Dairy-type productsinclude, for example, yogurt, cheese, ice cream, whipped topping, coffeecreamer, cream cheese, sour cream, cottage cheese, butter, mayonnaise,milk-based sauces, milk-based salad dressings, and cheese curds. Cerealgrain products include, for example, breads, muffins, bagels, pastries,noodles, cookies, pancakes, waffles, biscuits, semolina, chips,tortillas, cakes, crackers, breakfast cereals (including bothready-to-eat and cooked cereals), pretzels, dry bakery mixes, melbatoast, breadsticks, croutons, stuffing, energy bars, doughnuts, cakes,popcorn, taco shells, fry coatings, batters, breading, crusts, brownies,pies, puffed soy cakes, crepes, croissants, flour, and polenta. Saucecompositions include salad dressings, nut butter spreads (e.g., peanutbutter spreads), marinades, sauces, salsas, jams, cheese sauces,mayonnaise, tartar sauce, soy humus, dips, fruit syrups, and maplesyrups. Sauce composition may also include a suspending agent to aid inmaintaining the uniformity of the composition. Examples of suitablesuspending agents include polysaccharides, such as starch, cellulose(e.g., microcrystalline cellulose) and carrageenan, and polyuronides,such as pectin. Gelatin is another example of a suspending agent whichmay be used in the beverage compositions as well. Examples of additionalsupplemented food products prepared using the premixes in accordancewith the invention include tofu, formulated soy essence, powderedprotein supplements, juice mixable protein supplements, foaming agents,clouding agents, baby foods, meatless balls, meat analogues, eggproducts (e.g., scrambled eggs), soups, chowders, broth, milkalternatives, soy-milk products, chili, spice mixes, sprinkles, soywhiz, salad topping, edible films, edible sticks, chewing gum, baconbits, veggie bits, pizza crust barriers, soy pie, no-gas syntheticbeans, soy helper, soy cotton candy, fruit bits, pizza rolls, mashedpotatoes, spun soy protein fiber, soy roll-ups, extruded snacks,condiments, lotions, fries, gelatin dessert products, vitaminsupplements, nutritional bars, dry cake, bread or muffin mixes, andmicrowavable instant dry mixes.

In a particular aspect, the Chardonnay seed product may be provided asan energy bar (suitable for consumption during physical activity) or ameal replacement bar. The energy bar or meal replacement bar can alsocontain one or more vitamin, mineral, food supplement, botanical, orplant or herb extracts or ingredients known in the art or used in energybars or meal replacement bars, such as a fruit juice or extract, an herbor herb flavor, natural or artificial flavors, vitamins, minerals,anti-oxidant containing extracts, coenzyme Q, omega-3 fatty acids,guarana, caffeine, theobromine, maltodextrin, and protein. In someembodiments, the energy bar or meal replacement bar can have totalavailable energy levels of carbohydrates/protein/fat of 40/30/30respectively.

The energy and meal replacement bars can be further supplemented forathletic performance enhancement, mental energy or cognitive focusenhancement, and/or nutritional benefit. Exemplary supplements include,but are not limited to Vinpocetine, Vincamine Ginkgo Biloba, L-Arginine,Acetyl-L-Carnitine, Feverfew, DMAE (Dimethylaminoethanol), DMAEbitartrate, P-chlorophenoxyacetate, Vitamin B-Complex, Ginseng, 5 HTP(5-Hydroxytryptophan), L-Theanine, Androstenedione, L-Glutamine,L-Tyrosine, L-Glycine; L-lysine; Whey Protein; DHEA(Dehydroepiandrosterone).

In another aspect, the Chardonnay seed product may be provided in oradded to a liquid or powder infant formula. The infant formula cancontain a protein source, a fat source, and/or a carbohydrate source.The protein source can be, for example, dry or liquid cow's milk, wheyand/or casein, or soy protein. The fat source can be, for example, dairyfat and/or one or more vegetable oils. The carbohydrate source can be,for example, lactose, glucose, or sucrose. The infant formula canadditionally contain one or more vitamins, and/or one or more minerals.In an embodiment, the Chardonnay seed product may be added to acommercially available liquid or powder infant formula.

The Chardonnay seed composition can also contain an amount of a secondgrape seed or grape skin product which is not a Chardonnay seed product.In some embodiments, an amount of Chardonnay seed product is replaced inthe Chardonnay seed composition with an amount of the second grape seedor grape skin product. The amount of second grape seed or grape skinproduct that will need to be added to the Chardonnay seed composition toattain the same benefit as a given amount of Chardonnay seed product canreadily be determined by those skilled in the art.

4.4. Therapeutic Uses of Chardonnay Seed Flour Compositions

In accordance with the invention, a composition of the invention,comprising a Chardonnay seed flour composition, is administered to asubject, preferably a human subject, in which an increase in lipidmetabolism is useful or desired. The subject can be in need of treatmentor prevention of a cardiovascular disease, a dyslipidemia, a disorder ofglucose metabolism, Alzheimer's Disease, Syndrome X, a PPAR-associateddisorder, septicemia, a thrombotic disorder, obesity, pancreatitis,hypertension, a renal disease, cancer, inflammation, or impotence.

In another aspect of the invention, a composition of the invention,comprising a Chardonnay seed flour composition, is administered to asubject, preferably a human subject, in which modulation of the gutbiome is useful or desired. In an embodiment, the subject can be in needof treatment or prevention of, for example, lactic acidosis, colitis, orcolorectal cancer. In an embodiment, the subject is geneticallysusceptible to colon cancer.

In one embodiment, “treatment” or “treating” refers to an ameliorationof a disease or disorder, or at least one discernible symptom thereof.In another embodiment, “treatment” or “treating” refers to anamelioration of at least one measurable physical parameter, notnecessarily discernible by the subject. In yet another embodiment,“treatment” or “treating” refers to inhibiting the progression of adisease or disorder, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. In yet another embodiment, “treatment” or “treating”refers to delaying the onset of a disease or disorder.

In certain embodiments, the compositions of the invention areadministered to a subject, preferably a human, as a preventative measureagainst such diseases. As used herein, “prevention” or “preventing”refers to a reduction of the risk of acquiring a given disease ordisorder. In a preferred aspect, the compositions of the presentinvention are administered as a preventative measure to a subject,preferably a human having a genetic predisposition to a cardiovasculardisease, a dyslipidemia, a dyslipoproteinemia, a disorder of glucosemetabolism, Alzheimer's Disease, Syndrome X, a PPAR-associated disorder,septicemia, a thrombotic disorder, obesity, pancreatitis, hypertension,a renal disease, cancer, inflammation, or impotence. Examples of suchgenetic predispositions include but are not limited to the E4 allele ofapolipoprotein E, which increases the likelihood of Alzheimer's Disease;a loss of function or null mutation in the lipoprotein lipase genecoding region or promoter (e.g., mutations in the coding regionsresulting in the substitutions D9N and N291S; for a review of geneticmutations in the lipoprotein lipase gene that increase the risk ofcardiovascular diseases, dyslipidemias and dyslipoproteinemias, seeHayden and Ma, 1992, Mol. Cell Biochem. 113:171-176); and familialcombined hyperlipidemia and familial hypercholesterolemia.

In another preferred mode of the embodiment, the compositions of theinvention are administered as a preventative measure to a subject havinga non-genetic predisposition to a cardiovascular disease, adyslipidemia, a dyslipoproteinemia, a disorder of glucose metabolism,Alzheimer's Disease, Syndrome X, a PPAR-associated disorder, septicemia,a thrombotic disorder, obesity, pancreatitis, hypertension, a renaldisease, cancer, inflammation, or impotence. Examples of suchnon-genetic predispositions include but are not limited to cardiacbypass surgery and percutaneous transluminal coronary angioplasty, whichoften lead to restenosis, an accelerated form of atherosclerosis;diabetes in women, which often leads to polycystic ovarian disease; andcardiovascular disease, which often leads to impotence.

In certain embodiments, the mammal consumes a high-fat diet. In anembodiment, a high-fat diet is a diet in which at least 30%, 35%, or 40%of total daily calories are obtained from fat.

In certain embodiments, a second grape seed or grape skin product whichis not Chardonnay seed flour is administered to the mammal to provide aneffect which is greater than the effect of administration of Chardonnayseed flour alone.

4.4.1. Dyslipidemias

The present invention provides methods for the treatment or preventionof a dyslipidemia comprising administering to a subject atherapeutically effective amount of a Chardonnay seed flour composition.

As used herein, the term “dyslipidemias” refers to disorders that leadto or are manifested by aberrant levels of circulating lipids. To theextent that levels of lipids in the blood are too high, the compositionsof the invention are administered to a subject to restore normal levels.Normal levels of lipids are reported in medical treatises known to thoseof skill in the art. For example, recommended blood levels of LDL, HDL,free triglycerides and others parameters relating to lipid metabolismcan be found at the web sites of the American Heart Association and thatof the National Cholesterol Education Program of the National Heart,Lung and Blood Institute. At the present time, the recommended level ofHDL cholesterol in the blood is above 35 mg/dL; the recommended level ofLDL cholesterol in the blood is below 130 mg/dL; the recommended LDL:HDLcholesterol ratio in the blood is below 5:1, ideally 3.5:1; and therecommended level of free triglycerides in the blood is less than 200mg/dL.

Dyslipidemias which the Chardonnay seed flour compositions of thepresent invention are useful for preventing or treating include but arenot limited to hyperlipidemia and low blood levels of high densitylipoprotein (HDL) cholesterol. In certain embodiments, thehyperlipidemia for prevention or treatment by the compounds of thepresent invention is familial hypercholesterolemia; familial combinedhyperlipidemia; reduced or deficient lipoprotein lipase levels oractivity, including reductions or deficiencies resulting fromlipoprotein lipase mutations; hypertriglyceridemia;hypercholesterolemia; high blood levels of ketone bodies (e.g. n-OHbutyric acid); high blood levels of Lp(a) cholesterol; high blood levelsof low density lipoprotein (LDL) cholesterol; high blood levels of verylow density lipoprotein (VLDL) cholesterol and high blood levels ofnon-esterified fatty acids.

The present invention further provides methods for altering lipidmetabolism in a subject, e.g., reducing LDL in the blood of a subject,reducing free triglycerides in the blood of a subject, increasing theratio of HDL to LDL in the blood of a subject, and inhibiting saponifiedand/or non-saponified fatty acid synthesis, said methods comprisingadministering to the subject a Chardonnay seed flour composition in anamount effective alter lipid metabolism.

4.4.2. Cardiovascular Diseases

The present invention provides methods for the treatment or preventionof a cardiovascular disease, comprising administering to a subject atherapeutically effective amount of a Chardonnay seed flour composition.As used herein, the term “cardiovascular diseases” refers to diseases ofthe heart and circulatory system. These diseases are often associatedwith dyslipoproteinemias and/or dyslipidemias. Cardiovascular diseaseswhich the compositions of the present invention are useful forpreventing or treating include but are not limited to arteriosclerosis;atherosclerosis; stroke; ischemia; endothelium dysfunctions, inparticular those dysfunctions affecting blood vessel elasticity;peripheral vascular disease; coronary heart disease; myocardialinfarcation; cerebral infarction and restenosis.

4.4.3. Dyslipoproteinemias

The present invention provides methods for the treatment or preventionof a dyslipoproteinemia comprising administering to a subject atherapeutically effective amount of a Chardonnay seed flour composition.

As used herein, the term “dyslipoproteinemias” refers to disorders thatlead to or are manifested by aberrant levels of circulatinglipoproteins. To the extent that levels of lipoproteins in the blood aretoo high, the compositions of the invention are administered to asubject to restore normal levels. Conversely, to the extent that levelsof lipoproteins in the blood are too low, the compositions of theinvention are administered to a subject to restore normal levels. Normallevels of lipoproteins are reported in medical treatises known to thoseof skill in the art.

Dyslipoproteinemias which the compositions of the present invention areuseful for preventing or treating include but are not limited to highblood levels of LDL; high blood levels of apolipoprotein B (apo B); highblood levels of Lp(a); high blood levels of apo(a); high blood levels ofVLDL; low blood levels of HDL; reduced or deficient lipoprotein lipaselevels or activity, including reductions or deficiencies resulting fromlipoprotein lipase mutations; hypoalphalipoproteinemia; lipoproteinabnormalities associated with diabetes; lipoprotein abnormalitiesassociated with obesity; lipoprotein abnormalities associated withAlzheimer's Disease; and familial combined hyperlipidemia.

The present invention further provides methods for reducing apo C-Illevels in the blood of a subject; reducing apo C-III levels in the bloodof a subject; elevating the levels of HDL associated proteins, includingbut not limited to apo A-I, apo A-Il, apo A-IV and apo E in the blood ofa subject; elevating the levels of apo E in the blood of a subject, andpromoting clearance of triglycerides from the blood of a subject, saidmethods comprising administering to the subject a Chardonnay seed flourcomposition in an amount effective to bring about said reduction,elevation or promotion, respectively.

4.4.4. Glucose Metabolism Disorders

The present invention provides methods for the treatment or preventionof a glucose metabolism disorder, comprising administering to a subjecta therapeutically effective amount of a Chardonnay seed flourcomposition. As used herein, the term “glucose metabolism disorders”refers to disorders that lead to or are manifested by aberrant glucosestorage and/or utilization. To the extent that indicia of glucosemetabolism (i.e., blood insulin, blood glucose) are too high, thecompositions of the invention are administered to a subject to restorenormal levels. Conversely, to the extent that indicia of glucosemetabolism are too low, the compositions of the invention areadministered to a subject to restore normal levels. Normal indicia ofglucose metabolism are reported in medical treatises known to those ofskill in the art.

Glucose metabolism disorders which the compositions of the presentinvention are useful for preventing or treating include but are notlimited to impaired glucose tolerance; insulin resistance; insulinresistance related breast, colon or prostate cancer; diabetes, includingbut not limited to non-insulin dependent diabetes mellitus (NIDDM),insulin dependent diabetes mellitus (IDDM), gestational diabetesmellitus (GDM), and maturity onset diabetes of the young (MODY);pancreatitis; hypertension; polycystic ovarian disease; and high levelsof blood insulin and/or glucose.

The present invention further provides methods for altering glucosemetabolism in a subject, for example to increase insulin sensitivityand/or oxygen consumption of a subject, said methods comprisingadministering to the subject a Chardonnay seed flour composition in anamount effective to alter glucose metabolism.

4.4.5. Modulation of Gut Bacteria

Human gut biomes have been classified into three enterotypes accordingto the species dominating the bacterial population. These enterotypesare Bacteroides, Ruminococcus, and Prevotella. The Bacteroidesenterotype has been found to make several vitamins, including C and H,while the Prevotella enterotype has been found to make folic acid andvitamin B 1.

The present invention provides methods for modulating gut bacterialevels, comprising administering to a subject an amount of a Chardonnayseed flour composition effective to modulate gut bacteria levels. Asused herein, “modulate gut bacteria levels” refers to (i) a decrease inthe amount of total gut bacteria levels, and/or (ii) an increase or adecrease in bacteria levels of a subset of the total gut bacteria. Asubset of the total gut bacteria can be a single species, a genus, afamily, an order, a class, a phylum, or a combination of more than oneof the foregoing.

It may be desirable, for example, to modulate the levels of gut bacterialevels in infants born by cesarean section because bacterialcolonization of the gut is delayed in infants born by cesarean sectionas compared to infants born by vaginal delivery. Differences in gutmicrobiota can persist for up to six months after birth (Grolund, M. etal., Journal of Pediatric Gastroenterology & Nutrition, (1999), vol.28(1):19-25). For example, infants born by cesarean delivery aresignificantly less colonized with bacteria of the Bacteroides fragilisgroup than infants born by vaginal delivery at 6 months of age. Sevendays after birth, infants born by cesarean delivery have higher levelsof Enterobacteriaceae bacteria Citrobacter spp. and E. coli as apercentage of total gut bacteria compared to infants born by vaginaldelivery (Pandey, P. et al., J. Biosci. (2012) vol. 37(6):989-998).Chardonnay seed flour compositions can be used to increase the level ofBacteroides fragilis group bacteria and Clostridium spp., and decreasethe level of Enterobacteriaceae bacteria in infants born by cesareandelivery, for example, in order to promote the formation of a gut biomethat more closely resembles the gut microbiome of infants bornvaginally.

Chardonnay seed flour compositions can also be used to treat or preventlactic acidosis by reducing the amount of lactic acid producingbacteria, e.g., Lactobacillus spp., in the gut. Chardonnay seed flourcompositions can also be used to promote colonic health by increasingthe level of Clostridium spp. in the gut. Clostridium bacteria producebutyrate, which is the preferred energy source in the colonic mucosa.Butyrate protects against colitis and colorectal cancer, and isimportant for the normal development of colonic epithelial cells. Shen,J. et al., Applied and Environmental Microbiology, vol. 72:5232-5238(2006).

Chardonnay seed flour compositions can also be used to decrease levelsof Bifidobacterium spp. in the gut. However, if lowering the level ofBifidobacterium spp. is not desired but another therapeutic benefit ofChardonnay seed flour administration is desired, a probiotic supplementcontaining one or more Bifidobacterium species may be administered tothe subject to raise the levels of Bifidobacterium species in the gut ofthe subject. Examples of Bifidobacterium species that may beadministered in a probiotic supplement include Bifidobacterium bifidum,Bifidobacterium breve, and Bifidobacterium longum. The probioticsupplement can include additional bacterial species, for example, one ormore of Lactobacillus acidophilus, Lactobacillus plantarum,Lactobacillus salivarious, Lactobacillus casei, Lactobacilus paracasei,Lactobacillus rhamnosus, and Streptococcus thermophilus.

In an embodiment, the subject is identified as having an enterotypewhich is Bacteroides, Ruminococcus, or Prevotella. In an embodiment, thesubject has a Bacteroides enterotype. In an embodiment, the subject hasa Ruminococcus enterotype. In an embodiment, the subject has aPrevotella enterotype.

4.5. Therapeutic Uses of Chardonnay Seed Extract Compositions

In accordance with the invention, a composition of the invention,comprising a Chardonnay seed extract composition, is administered to asubject, preferably a human subject, for each of the therapeutic usesdescribed in Section 4.4. Chardonnay seed extract has similar biologicaleffects as Chardonnay seed flour (data not shown). In order to achieve abenefit which in comparable to the benefit obtained by using an amountof Chardonnay seed flour, the amount of Chardonnay seed extractadministered should be an amount which provides 2-5 times the catechinlevels of the amount of Chardonnay seed flour. Specific amounts ofChardonnay seed extract that will need to be consumed daily to attainthe same benefit as a given amount of Chardonnay seed flour can readilybe determined by those skilled in the art.

5. EXAMPLE 1: COMPARISON OF HEALTH BENEFITS OF CHARDONNAY, SAUVIGNONBLANC AND WHITE RIESLING PRODUCTS

5.1. Materials & Methods

5.1.1. Hamsters and Diets

Male Golden Syrian hamsters (˜80 g, LVG strain, Charles River) wereacclimatized and given water and a 5001 rodent diet (LabDiet, PMIInternational; protein, 239 g/kg; fat, 50 g/kg; non-nitrogenoussubstances, 487 g/kg; crude fiber, 51 g/kg; ash, 70 g/kg; energy, 17MJ/kg; and sufficient amounts of minerals and vitamins for healthymaintenance) ad libitum for 1 week prior to the initiation of theexperimental diets. Hamsters were weighed and randomized into 2 groupsof 15 hamsters each and were fed high-fat diets ad libitum containingeither 10% (by weight) Chardonnay, Cabernet or Syrah grape seed flour orgrape skin flour or the control (Diets) for 4 weeks. Grapes from whichthe flours were produced were grown in the coastal valleys of NorthernCalifornia. The compositions of the flours are shown in FIG. 1. Dietsconsisted of 18% of energy as protein, 43% as carbohydrate, and 39% asfat supplemented with 0.1% cholesterol (FIG. 2) GenOil refers to acommercially available grape seed oil from an unspecified grape variety.Body weights were recorded weekly and food intake was monitored twiceper week. The study was approved by the Animal Care and Use Committee,Western Regional Research Center, USDA, Albany, Calif.

5.1.2. Plasma and Tissue Collection

Hamsters were fasted for 12 hours and anesthetized with Isoflurane(Phoenix Pharmaceutical). Blood was collected by cardiac puncture withsyringes previously rinsed with potassium EDTA solution (15% wt:v) andplasma was separated after centrifugation at 2000×g for 30 minutes at 4°C. Livers were collected, weighed, and immediately frozen in liquidnitrogen for analysis.

5.1.3. Plasma Biomarker Analysis

Cholesterol in plasma lipoproteins were determined by size-exclusionchromatography as previously described (German et al., 1996. Nutr Res.1996; 16:1239-49). Plasma triglycerides, total cholesterol, freecholesterol, and glucose were determined by enzymatic colorimetricassays using a Roche Diagnostics/Hitachi 914 Clinical Analyzer withassay kits (Roche Diagnostics and Wako Chemicals). Plasma concentrationsof adiponectin (B-bridge International) and insulin (Mercodia) offeed-deprived hamsters were determined using mouse adiponectin andultra-sensitive rat insulin immunoassay kits as previously described(Hung et al., 2009, J. Diabetes 1:194-206). Blood glucose concentrationsin feed-deprived hamsters were measured in tail vein samples using aOneTouch Ultrameter (LifeScan).

5.1.4. Hepatic Lipid Analysis

Lyophilized, ground liver samples were extracted using an acceleratedsolvent extractor (Dionex) at 100° C.; 13.8 MPa with 75/25hexane/2-propanol. The sample extract was analyzed on a RocheDiagnostic/Hitachi 914 clinical analyzer (Roche Diagnostics) todetermine hepatic triglycerides, total cholesterol, and free cholesterolusing the kits described above.

5.1.5. Fecal Bile Acids and Sterol Analysis

Feces were collected for 3 consecutive days immediately prior to whenthe hamsters were killed and were lyophilized, milled, and stored at−20° C. Bile acids and sterols were determined by HPLC as describedpreviously (Hong et al., 2007, J Agric Food Chem. 55:9750-7).

5.1.6. Statistical Analysis

All data are expressed as means±SE. Differences between control anddifferent diet groups were determined by 2-tailed Student's t tests.When variances of each group were unequal, significance of differenceswas determined using the Welch's test. Pearson correlation coefficientswere calculated for investigating relationships of plasma totalcholesterol, plasma adiponectin concentrations, hepatic cholesterol, andtriglyceride concentrations with the expression of hepatic genes (JMP 7statistical program, SAS Institute). Significance was defined at P<0.05.

5.2. Results

The animals fed Chardonnay seed flour had significantly decreased weightcompared to animals fed the other diets (FIGS. 3-4). Animals fed theChardonnay seed flour diet had an average weight of about 97 grams afterfour weeks. Animals fed the control diet had an average weight of about110 grams after four weeks.

Although the Chardonnay seed flour treatment animals weighed less thancontrol animals, the Chardonnay seed flour treatment animals ate more(FIG. 5), both in terms of volume (FIG. 6) and calories (FIG. 7).

Animals fed the Chardonnay seed flour diet showed the greatest decreasein VLDL (66%) and LDL (50%) cholesterol (FIG. 8). Blood glucose showed aslight reduction (FIG. 9).

Chardonnay seed flour lowered liver weights and epididimal adiposetissue (EA) weight 30% and 20%, respectively (FIG. 10).

These data show that Chardonnay seed flour was the obvious standoutamong grape products tested. Chardonnay skin flour showed a similartrend but not as great a benefit as Chardonnay seed flour.

6. EXAMPLE 2: COMPARISON OF HEALTH BENEFITS OF CHARDONNAY, SAUVIGNONBLANC AND WHITE RIESLING PRODUCTS

6.1. Materials & Methods

6.1.1. Hamsters and Diets

Male Golden Syrian hamsters (˜80 g, LVG strain, Charles River) wereacclimatized and given water and a 5001 rodent diet (LabDiet, PMIInternational; protein, 239 g/kg; fat, 50 g/kg; non-nitrogenoussubstances, 487 g/kg; crude fiber, 51 g/kg; ash, 70 g/kg; energy, 17MJ/kg; and sufficient amounts of minerals and vitamins for healthymaintenance) ad libitum for 1 week prior to the initiation of theexperimental diets. Hamsters were weighed and randomized into 10 groupsof 10 hamsters each and were fed high-fat diets ad libitum containingChardonnay seed ethanol extract, Chardonnay seed methanol extract,Chardonnay seed ethanol extracted residue, Chardonnay seed methanolextracted residue, 10% (by weight) Chardonnay seed flour, 10% (byweight) Sauvignon Blanc seed flour, 10% (by weight) White Riesling seedflour, 7% (by weight) Chardonnay seed flour, 3% (by weight) Chardonnayseed flour, or a control diet for 4 weeks. Grapes from which the flourswere produced were grown in the coastal valleys of Northern California.Ethanol extracts and extracted residues were prepared by a “teaextraction method” comprising the following steps: (1) solvent (1625 mlof 40% ethanol in distilled water) was added to 325 g Chardonnay seedflour and shaken at 80° C. for two hours; (2) the mixture from step (1)was then filtered through Whatman 1 filter paper; (3) ethanol was thenremoved from the filtrate from step (2) on a rotovac; (4) the solutionfrom step (3) and filter cake from step (2) were frozen and freeze driedto produce Chardonnay seed ethanol extract and Chardonnay seed ethanolextracted residue, respectively. Similar procedures were used to produceChardonnay seed methanol extract and Chardonnay seed methanol extractedresidue.

Diets consisted of 18% of energy as protein, 43% as carbohydrate, and39% as fat supplemented with 0.1% cholesterol. Body weights wererecorded weekly and food intake was monitored twice per week. The studywas approved by the Animal Care and Use Committee, Western RegionalResearch Center, USDA, Albany, Calif.

6.1.2. Plasma and Tissue Collection

Hamsters were fasted for 12 hours and anesthetized with Isoflurane(Phoenix Pharmaceutical). Blood was collected by cardiac puncture withsyringes previously rinsed with potassium EDTA solution (15% wt:v) andplasma was separated after centrifugation at 2000×g for 30 min at 4° C.Livers were collected, weighed, and immediately frozen in liquidnitrogen for analysis.

6.1.3. Plasma Biomarker Analysis

Cholesterol in plasma lipoproteins were determined by size-exclusionchromatography as previously described (German et al., 1996, Nutr Res.16:1239-49). Plasma triglycerides, total cholesterol, free cholesterol,and glucose were determined by enzymatic colorimetric assays using aRoche Diagnostics/Hitachi 914 Clinical Analyzer with assay kits (RocheDiagnostics and Wako Chemicals). Plasma concentrations of adiponectin(B-bridge International) and insulin (Mercodia) of feed-deprivedhamsters were determined using mouse adiponectin and ultra-sensitive ratinsulin immunoassay kits as previously described (Hung et al., 2009, JDiabetes. 1:194-206). Blood glucose concentrations in feed-deprivedhamsters were measured in tail vein samples using a OneTouch Ultrameter(LifeScan).

6.1.4. Hepatic Lipid Analysis

Lyophilized, ground liver samples were extracted using an acceleratedsolvent extractor (Dionex) at 100° C.; 13.8 MPa with 75/25hexane/2-propanol. The sample extract was analyzed on a RocheDiagnostic/Hitachi 914 clinical analyzer (Roche Diagnostics) todetermine hepatic triglycerides, total cholesterol, and free cholesterolusing the kits described above.

6.1.5. Fecal Bile Acids and Sterol Analysis

Feces were collected for 3 consecutive days immediately prior to whenthe hamsters were killed and were lyophilized, milled, and stored at−20° C. Bile acids and sterols were determined by HPLC as describedpreviously (Hong et al., 2007, Agric Food Chem. 55:9750-7).

6.1.6. Statistical Analysis

All data are expressed as means±SE. Differences between control anddifferent diet groups were determined by 2-tailed Student's t tests.When variances of each group were unequal, significance of differenceswas determined using the Welch's test. Pearson correlation coefficientswere calculated for investigating relationships of plasma totalcholesterol, plasma adiponectin concentrations, hepatic cholesterol, andtriglyceride concentrations with the expression of hepatic genes (JMP 7statistical program, SAS Institute). Significance was defined at P<0.05.

6.2. Results

Chardonnay seed flour at 10% by weight of the diet showed reproducibleresults with Example 1. Animals fed the diet with Chardonnay seed flourat 10% by weight of the diet had the least weight gain on a 35% fat dietcompared to the control animals.

Chardonnay seed flour showed a dose-response. Animals were fed 10%, 7%or 3% Chardonnay seed flour as part of their diet for 4 weeks.Increasing response, indicated as weight difference, was observed; themore the animals ate the less weight they added (FIG. 12).

Chardonnay seed extract showed some effect on weight but not as dramaticas the direct addition of Chardonnay seed flour.

Chardonnay seed extracts and Chardonnay seed extract residues showedsome effect on cholesterol levels (FIG. 13) and LDL/HDL ratio (FIG. 14),but not as dramatic as the direct addition of Chardonnay seed flour.Similarly, the Chardonnay seed extracts showed some effect on organweight (FIG. 15), but not as dramatic as the direct addition ofChardonnay seed flour.

Other white seed flours; e.g. Sauvignon Blanc or White Riesling showedlittle to no difference in weights compared to the control diets.Chardonnay has been compared side by side with four other varietals andis a clear standout.

7. EXAMPLE 3: EFFECTS OF CHARDONNAY SEED FLOUR ON RELATIVE ADIPOSE ANDHEPATIC GENE EXPRESSION

7.1. Materials & Methods

7.1.1. Hamsters and Diets

Male Golden Syrian hamsters (˜80 g, LVG strain, Charles River) wereacclimatized and given water and a 5001 rodent diet (LabDiet, PMIInternational; protein, 239 g/kg; fat, 50 g/kg; non-nitrogenoussubstances, 487 g/kg; crude fiber, 51 g/kg; ash, 70 g/kg; energy, 17MJ/kg; and sufficient amounts of minerals and vitamins for healthymaintenance) ad libitum for 1 week prior to the initiation of theexperimental diets. Hamsters were weighed and randomized into 2 groupsand fed high-fat diets ad libitum containing either 10% (by weight)Chardonnay grape seed flour or a control diet for 4 weeks. Grapes fromwhich the flours were produced were grown in the coastal valleys ofNorthern California. Diets consisted of 18% of energy as protein, 43% ascarbohydrate, and 39% as fat supplemented with 0.1% cholesterol. Bodyweights were recorded weekly and food intake was monitored twice perweek. The study was approved by the Animal Care and Use Committee,Western Regional Research Center, USDA, Albany, Calif.

7.1.2. mRNA Analysis

Quantitative PCR (qPCR) was used to measure mRNA expression of selectedgenes of the inflammation, cholesterol, bile acid, and fatty acidpathways in adipose and liver samples from hamsters fed either theChardonnay seed flour supplemented diet or the control diet.

7.1.3. Statistical Analysis

All data are expressed as averages±SD. Averages shown are relativevalues comparing mRNA levels in adipose or liver tissue from hamstersfed a diet supplemented with Chardonnay seed flour to mRNA levels inadipose or liver tissue from hamsters fed the control diet.

7.2. Results

Relative gene expression levels comparing gene expression in adiposetissue from hamsters fed a diet supplemented with Chardonnay seed flourto gene expression in adipose tissue from hamsters fed the control dietare shown in FIG. 16. Relative gene expression levels comparing geneexpression in hepatic tissue from hamsters fed a diet supplemented withChardonnay seed flour to gene expression in hepatic tissue from hamstersfed the control diet are shown in FIG. 17.

Hepatic ACOX1, CYP51, and CYP7a1 gene expression was notably elevated inhamsters fed the Chardonnay seed flour supplemented diet, while hepaticABCG5 and SCD1 gene expression was notably decreased in hamsters fed theChardonnay seed flour supplemented diet.

ACOX1 is involved in the regulation of fat oxidation, CYP51 is involvedin the regulation of cholesterol biosynthesis, CYP7a1 is involved in theregulation of bile acid synthesis, SCD1 is involved in fat synthesis,and ABCG5 is involved in the transport of cholesterol back into theintestine. Accordingly, the results suggest that a diet supplementedwith Chardonnay seed flour may reduce cholesterol and/or bilere-absorption, leading to reduced levels in the liver. The resultsfurther suggest that a diet supplemented with Chardonnay seed flour mayreduce weight gain by hepatic gene up-regulation for fat oxidation(ACOX1) and/or by down-regulation of fat synthesis (SCD1).

8. EXAMPLE 4: COMPARISON OF HEALTH BENEFITS OF CHARDONNAY SEED FLOUR ANDCHARDONNAY GRAPE SEED EXTRACT TO COMMERCIALLY AVAILABLE GRAPE SEEDEXTRACTS

8.1. Materials & Methods

8.1.1. Hamsters and Diets

Male Golden Syrian hamsters (˜80 g, LVG strain, Charles River) wereacclimatized and given water and a 5001 rodent diet (LabDiet, PMIInternational; protein, 239 g/kg; fat, 50 g/kg; non-nitrogenoussubstances, 487 g/kg; crude fiber, 51 g/kg; ash, 70 g/kg; energy, 17MJ/kg; and sufficient amounts of minerals and vitamins for healthymaintenance) ad libitum for 1 week prior to the initiation of theexperimental diets. All grape seed extracts were characterized by HPLCmethods and their feeding amounts were adjusted to provide the sameamount of catechin as a 7% (w/w) Chardonnay seed flour. Hamsters wereweighed and randomized into 10 groups of 8 hamsters each and were fedhigh-fat diets ad libitum containing 7% (by weight) Chardonnay seedflour (1 group), Vitacost® grape seed extract (2 groups), Mega Natural®BP grape seed extract (2 groups), Leucoselect® grape seed extract (2groups), catechin (approximately 0.000785 g/g diet) (1 group),epicatechin (approximately 0.00104 g/g diet) (1 group), or a controldiet (1 group) for 4 weeks. Catechin levels were used to normalize eachgrape seed extract to the amount of Chardonnay seed flour. Epicatechinand catechin were included in the chow diet as controls for thesecompounds, which were implicated in other studies to be responsible forblood pressure and lipid regulation. Grapes from which the Chardonnayseed flour was produced were grown in the coastal valleys of NorthernCalifornia. Diets consisted of 18% of energy as protein, 43% ascarbohydrate, and 39% as fat supplemented with 0.1% cholesterol. Bodyweights were recorded weekly and food intake was monitored twice perweek. The study was approved by the Animal Care and Use Committee,Western Regional Research Center, USDA, Albany, Calif.

8.1.2. Plasma and Tissue Collection

Hamsters were fasted for 12 hours and anesthetized with Isoflurane(Phoenix Pharmaceutical). Blood was collected by cardiac puncture withsyringes previously rinsed with potassium EDTA solution (15% wt:v) andplasma was separated after centrifugation at 2000×g for 30 minutes at 4°C. Livers were collected, weighed, and immediately frozen in liquidnitrogen for analysis.

8.1.3. Plasma Biomarker Analysis

Cholesterol in plasma lipoproteins were determined by size-exclusionchromatography as previously described (German et al., 1996. Nutr Res.1996; 16:1239-49). Plasma triglycerides, total cholesterol, freecholesterol, and glucose were determined by enzymatic colorimetricassays using a Roche Diagnostics/Hitachi 914 Clinical Analyzer withassay kits (Roche Diagnostics and Wako Chemicals). Plasma concentrationsof adiponectin (B-bridge International) and insulin (Mercodia) offeed-deprived hamsters were determined using mouse adiponectin andultra-sensitive rat insulin immunoassay kits as previously described(Hung et al., 2009, J. Diabetes 1:194-206). Blood glucose concentrationsin feed-deprived hamsters were measured in tail vein samples using aOneTouch Ultrameter (LifeScan).

8.1.4. Hepatic Lipid Analysis

Lyophilized, ground liver samples were extracted using an acceleratedsolvent extractor (Dionex) at 100° C.; 13.8 MPa with 75/25hexane/2-propanol. The sample extract was analyzed on a RocheDiagnostic/Hitachi 914 clinical analyzer (Roche Diagnostics) todetermine hepatic triglycerides, total cholesterol, and free cholesterolusing the kits described above.

8.1.5. Fecal Bile Acids and Sterol Analysis

Feces were collected for 3 consecutive days immediately prior to whenthe hamsters were killed and were lyophilized, milled, and stored at−20° C. Bile acids and sterols were determined by HPLC as describedpreviously (Hong et al., 2007, J Agric Food Chem. 55:9750-7).

8.1.6. Statistical Analysis

All data are expressed as means+SE. Differences between control anddifferent diet groups were determined by 2-tailed. Student's t tests.When variances of each group were unequal, significance of differenceswas determined using the Welch's test. Pearson correlation coefficientswere calculated for investigating relationships of plasma totalcholesterol, plasma adiponectin concentrations, hepatic cholesterol, andtriglyceride concentrations with the expression of hepatic genes (JMP 7statistical program, SAS Institute). Significance was defined at P<0.05.

8.2. Results

The animals fed Chardonnay seed flour had significantly decreased plasmalevels of total cholesterol and LDL cholesterol compared to animals feddiets supplemented with commercially available grape seed extracts(FIGS. 18-19), with the exception of one of the groups fed a dietsupplemented with Leucoselect® grape seed extract (L7). These data showthat Chardonnay seed flour was superior to all but one of the commercialgrape seed extracts tested for lowering total cholesterol and LDLcholesterol.

9. EXAMPLE 5: EFFECTS OF CHARDONNAY SEED FLOUR ON FECAL MICROBIOTALEVELS

9.1. Materials & Methods

9.1.1. Fecal Samples

Feces were collected from the animals of Example 1 at day 0 (control)and at day 20 (Chardonnay seed flour supplemented diet, Cabernet seedflour supplemented diet, and high-fat control diet). The feces weredried and frozen.

9.1.2. RNA Extraction and Quantification

Frozen fecal samples were added to 10 volumes of RNAlater®-ICE (AppliedBiosystems, Foster City, Calif., USA) for at least 24 hours. Total RNAwas prepared using the Stool Total RNA Purification Kit (Norgen BiotekCorp., Canada) following the manufacturer's protocols.

Quantity and purity of the isolated RNA was confirmed byspectrophotometry (A260/A280 ratio). cDNA was prepared for each sampleusing 250 ng of total RNA and a PrimeScript™ RT reagent kit (Takara BioInc., Shiga, Japan) according to the manufacturer's protocols.

Real-time PCR for quantification of intestinal bacterial 16S rRNA geneexpression was performed using the AB 7500 Realtime PCR system (AppliedBiosystems, Foster City, Calif., USA). Amplification was performed induplicate using SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan).Amplifications using the primers shown in Table 1 were performed withthe following temperature profiles: one cycle at 95° C. for 30 seconds,and 40 cycles of denaturation at 95° C. for 5 to 10 seconds, annealatingat the optimal temperature for 5 to 15 seconds and elongation at 72° C.for 20 seconds.

TABLE 1 Bacterial Strains SEQ Target Primer Sequence (5′ to 3′)for standard curve ID NO Lactobacillus Forward AGCAGTAGGGAATCTTCCALactobacillus delbrueckii  1 spp. subsp. bulgaricus (KCTC 3635) ReverseCACCGCTACACATGGAG  2 Bifidobacterium Forward CTCCTGGAAACGGGTGGBifidobacterium animalis  3 spp. subsp. lactis (KCTC 5854) ReverseGGTGTTCTTCCCGATATCTACA  4 Enterococcus Forward CCCTTATTGTTAGTTGCCATCATTEnterococcus faecalis  5 spp. (KCTC 3206) Reverse ACTCGTTGTACTTCCCATTGT 6 Clostridium Forward GCACAAGCAGTGGAGT Clostridium leptum  7 cluster IV(KCTC 5155) Reverse AGTSCTCTTGGGTAG  8 Pseudomonas ForwardCAAGCCCTACAAGAAATCCG Pseudomonas aeruginosa  9 aeruginosa (KCTC 1636)Reverse TCCACCGAACCGAAGTTG 10 Staphylococcus ForwardGCGATTGATGGTGATACGGTT Staphylococcus aureus 11 aureus (ATCC 6538)Reverse AGCCAAGCCTTGACGAACTAAAGC 12 Total bacteria ForwardTCCTACGGGAGGCAGCAGT Escherichia coli 13 (KCTC 1682) ReverseGGACTACCAGGGTATCTAATCCTGTT 14

9.2. Results

The results of quantitative real-time PCR of the fecal samples of thehamsters fed a high fat diet containing Chardonnay or Cabernet seedflour revealed that the intestinal microbiota was modulated by theconsumption of the flours. Measured total bacteria levels in fecalmatter from hamsters fed the four diets are shown in FIG. 20. Measuredlevels of bacterial subpopulations are shown in FIGS. 21-26.

Fecal samples from hamsters fed a high-fat diet supplemented withChardonnay seed flour showed significantly reduced levels ofBifidobacterium spp., Lactobacillus spp., and Enterobacteriaceae, andsignificantly increased levels of Enterococcus spp., bacteria ofClostridium cluster IV and the Bacteroides fragilis group compared tofecal samples from hamsters fed the high-fat control diet.

Fecal samples from hamsters fed a high-fat diet supplemented withCabernet seed flour showed significantly reduced levels ofEnterobacteriaceae, and significantly increased levels of Enterococcusspp., and the Bacteroides fragilis group compared to fecal samples fromhamsters fed the high-fat control diet.

10. SPECIFIC EMBODIMENTS AND INCORPORATION BY REFERENCE

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A method of treating metabolic syndrome in amammal in need thereof comprising administering to the mammal in needthereof an effective amount of Chardonnay pomace meal.
 2. The method ofclaim 1, wherein the treating metabolic syndrome reduces cholesterol ofthe mammal.
 3. The method of claim 1, wherein the treating metabolicsyndrome reduces the weight of the mammal.
 4. The method of claim 1,wherein the treating metabolic syndrome reduces the weight of the liverof the mammal.
 5. The method of claim 1, wherein the treating metabolicsyndrome restores blood glucose levels to normal in the mammal.
 6. Themethod of claim 1, wherein the mammal is a human.
 7. A method ofreducing the weight of a liver or reducing cholesterol in a mammal inneed thereof comprising administering to the mammal in need thereof aneffective amount of cold or hot pressed dried Chardonnay seed flour andChardonnay pomace meal.
 8. The method of claim 7, wherein the methodcomprises reducing the weight of the liver in the mammal.
 9. The methodof claim 7, wherein the method comprises reducing cholesterol in themammal.
 10. The method of claim 7, wherein the mammal is a human. 11.The method of claim 7, wherein the method comprises administering aneffective amount of Chardonnay pomace meal to the mammal.
 12. A methodof treating diabetes, increasing lipid metabolism, reducing weight,treating dyslipoproteinemia, increasing the amount of Bacteroidesfragilis in the gut, decreasing the amount of Enterobacteriaceaebacteria in the gut, increasing the amount of Clostridium bacteria inthe gut, or treating lactic acidosis in a mammal in need thereof, themethod comprising administering to the mammal in need thereof aneffective amount of Chardonnay pomace meal.
 13. The method of claim 12,wherein the mammal is a human.
 14. The method of claim 12, wherein theeffective amount is at least 0.2 g/kg of body weight is administered tothe mammal.
 15. The method of claim 12, wherein the effective amount isat least 1.0 g per day is administered to the mammal.
 16. The method ofclaim 12, wherein the mammal is a human.